[Atorvastatin promotes the healing of alveolar bone defect in rats and its effect on Wnt/ß-catenin signaling pathway].

PURPOSE: To investigate the therapeutic effect of atorvastatin on alveolar bone defect model in rats, and to observe the effect of atorvastatin on Wnt/ß-catenin. METHODS: Thirty rats were randomly divided into normal group (group N), model group (group M) and atorvastatin administration group (group ATV). Except group N, bone defects were made in other rats' alveolar bone to construct alveolar bone defect model. After successful modeling, 20 mg/kg atorvastatin suspension was administered by gavage in group ATV, and the same amount of sodium carboxymethyl cellulose solution was administered by gavage in group N and group M for twenty-one days. After the last administration, tail vein blood was collected to detect the concentrations of serum osteoprotegerin (OPG), alkaline phosphatase (ALP) and osteocalcin (BPG). H-E staining was used to observe the pathological changes of maxillary defect area, and lane Sandhu score was performed. Tartrate resistant acid phosphatase(TRAP) staining was used to detect the number of osteoclasts in the defect area. Real time fluorescence quantitative PCR(RT-qPCR) and Western blot(WB) were used to detect Wnt, ß-catenin and Runx2 mRNA protein expression. Statistical analysis was performed with SPSS 23.0 software package. RESULTS: Compared with group N, the concentrations of OPG, ALP, BGP and Lane Sandhu score in group M decreased, and the number of osteoclasts increased. Compared with group M, the concentrations of OPG, ALP and BGP and lane Sandhu score in group ATV increased, and the number of osteoclasts decreased. After H-E staining, the amount of bone formation in maxillary defect area in group N was more,there was fewer bone tissues in the defect area in group M, the amount of bone tissues in the defect area increased in group ATV. Compared with group N, Wnt, ß-catenin and Runx2 mRNA protein decreased. Compared with group M, Wnt, ß-catenin and Runx2 mRNA protein expression increased. CONCLUSIONS: Atorvastatin can promote the healing of alveolar bone defect and accelerate bone reconstruction in rat models. This effect may be related to the activation of Wnt/ß-catenin signaling pathway.

Aspirin Stimulates the Osteogenic Differentiation of Human Adipose Tissue-Derived Stem Cells In Vitro.

This study investigates the impact of acetylsalicylic acid (ASA), also known as aspirin, on adipose tissue-derived stem cells (ASCs), aiming to elucidate its dose-dependent effects on morphology, viability, proliferation, and osteogenic differentiation. Isolated and characterized human ASCs were exposed to 0 µM, 100 µM, 200 µM, 400 µM, 800 µM, 1000 µM, 10,000 µM, and 16,000 µM of ASA in vitro. Cell morphology, viability, and proliferation were evaluated with fluorescent live/dead staining, alamarBlue viability reagent, and CyQUANT® cell proliferation assay, respectively. Osteogenic differentiation under stimulation with 400 µM or 1000 µM of ASA was assessed with alizarin red staining and qPCR of selected osteogenic differentiation markers (RUNX2, SPP1, ALPL, BGLAP) over a 3- and 21-day-period. ASA doses ≤ 1000 µM showed no significant impact on cell viability and proliferation. Live/dead staining revealed a visible reduction in viable cell confluency for ASA concentrations ≥ 1000 µM. Doses of 10,000 µM and 16,000 µM of ASA exhibited a strong cytotoxic and anti-proliferative effect in ASCs. Alizarin red staining revealed enhanced calcium accretion under the influence of ASA, which was macro- and microscopically visible and significant for 1000 µM of ASA (p = 0.0092) in quantification if compared to osteogenic differentiation without ASA addition over a 21-day-period. This enhancement correlated with a more pronounced upregulation of osteogenic markers under ASA exposure (ns). Our results indicate a stimulatory effect of 1000 µM of ASA on the osteogenic differentiation of ASCs. Further research is needed to elucidate the precise molecular mechanisms underlying this effect; however, this discovery suggests promising opportunities for enhancing bone tissue engineering with ASCs as cell source.

[Effect and mechanism of Gusong Qianggu Decoction on reducing bone loss in ovariectomized mice by targeting endoplasmic reticulum stress-induced apoptosis of osteocytes].

This study aims to investigate the role and mechanism of Gusong Qianggu Decoction(GSQG) in attenuating bone loss in ovariectomized mice by targeting the endoplasmic reticulum stress(ERS)-induced apoptosis of osteocytes. After the modeling of osteoporosis in mice with bilateral ovary removal(OVX), 60 mice were randomized by the random number method into six groups: sham,model, low-, medium-, and high-dose GSQG(GSQG-L, GSQG-M, and GSQG-H, respectively), and estradiol(E_2), with 10 mice in each group. The mice in each group were administrated with corresponding drugs by gavage one month after surgery and the administration lasted for 3 months. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the serum levels of osteocalcin(OCN), procollagen type Ⅰ N-terminal propeptide(PINP), carboxy-terminal cross-linked telopeptide of type Ⅰ collagen(CTX),and anti-tartarte acid phosphatase 5b(TRAcP-5b). Micro-CT was employed to observe the changes in bone microstructure of the distal femur. Hematoxylin-eosin(HE) staining was employed to observe the morphology of the bone tissue. RT-qPCR was conducted to determine the m RNA levels of tibial stem osteogenesis-associated genes [type Ⅰ collagen(Col-Ⅰ), alkaline phosphatase(ALP), Runtrelated transcription factor-2(Runx2), bone sialoprotein(BSP), and OCN] and bone-breaking related genes [tartrate-resistant acid phosphatase(TRAP), nuclear factor-activated T cell 1(NFATc1), and cathepsin K(CATK)]. TUNEL staining and immunohistochemistry were employed to detect the apoptosis of osteoblasts. Western blot was employed to measure the expression of ERS-related proteins glucose-regulated protein 78( Grp78), protein kinase RNA-like endoplasmic reticulum kinase( PERK), phosphorylated PERK(p-PERK),eukaryotic translation initiation factor 2 alpha(eIF2α), phosphorylated e IF2α(p-eIF2α), inositol-requiring enzyme 1 alpha(IRE1α), phosphorylated IRE1α(p-IRE1α), and activating transcription factor 6(ATF6) in the proximal tibial bone tissue. The results showed that GSQG significantly recovered the levels of OCN, PINP, TRAc P-5b, and CTX in the serum of ovariectomized mice, and Micro-CT showed that GSQG improved the bone microstructure of distal femur in a dose-dependent manner. Compared with the model group, GSQG widened and increased the bone trabeculae, restored the reticular structure with neat arrangement and enlarged interstitial gaps, and reduced the number of TUNEL-positive cells(P<0. 05, P<0. 01). Furthermore, GSQG down-regulated the expression levels of cysteine aspartate protease-3( caspase-3) and factor Bcl-2-associated X protein( Bax)(P< 0. 05,P<0. 01) and up-regulated the expression level of Bcl-2(P<0. 05, P<0. 01). The GSQG groups showed up-regulated m RNA levels of Col-Ⅰ, ALP, Runx2, BSP, and OCN(P< 0. 01) and down-regulated m RNA levels of TRAP, NFATc1, and CATK(P< 0. 05,P<0. 01). In addition, GSQG, especially GSQG-H, down-regulated the protein levels of Grp78, p-PERK, p-eIF2, p-IRE1α, and ATF6(P< 0. 05, P< 0. 01). In conclusion, GSQG can inhibit the apoptosis of osteocytes by inhibiting the Grp78/PERK/e IF2α/IRE1α/ATF6 signaling pathway in the proximal tibia tissue, thus reducing bone loss in ovariectomized mice.

Exposure to BPA and BPS during pregnancy disrupts the bone mineralization in the offspring.

Exposure to plastic-derived estrogen-mimicking endocrine-disrupting bisphenols can have a long-lasting effect on bone health. However, gestational exposure to bisphenol A (BPA) and its analogue, bisphenol S (BPS), on offspring's bone mineralization is unclear. The effects of in-utero bisphenol exposure were examined on the offspring's bone parameters. BPA and BPS (0.0, 0.4 µg/kg bw) were administered to pregnant Wistar rats via oral gavage from gestational day 4-21. Maternal exposure to BPA and BPS increased bone mineral content and density in the offspring aged 30 and 90 days (P < 0.05). Plasma analysis revealed that alkaline phosphatase, and Gla-type osteocalcin were significantly elevated in the BPS-exposed offspring (P < 0.05). The expression of BMP1, BMP4, and their signaling mediators SMAD1 mRNAs were decreased in BPS-exposed osteoblast SaOS-2 cells (P < 0.05). The expression of extracellular matrix proteins such as ALPL, COL1A1, DMP1, and FN1 were downregulated (P < 0.05). Bisphenol co-incubation with noggin decreased TGF-ß1 expression, indicating its involvement in bone mineralization. Altered mineralization could be due to dysregulated expression of bone morphogenetic proteins and signalling mediators in the osteoblast cells. Thus, bisphenol exposure during gestation altered growth and bone mineralization in the offspring, possibly by modulating the expression of Smad-dependent BMP/TGF-ß1 signalling mediators.

Enhanced osteogenic differentiation potential of Arnica montana and Bellis perennis in C3H10T1/2 multipotent mesenchymal stem cells.

BACKGROUND: Arnica montana and Bellis perennis are two medicinal plants that are thought to accelerate bone repair in homoeopathic literature. Mesenchymal stem cells (MSCs) are multipotent stem cells with the ability to differentiate and regenerate bone or osteogenesis. Hence, we aimed to determine the role of Arnica montana and Bellis perennis on the osteogenic differentiation of the C3H10T1/2 stem cell line. METHODS AND RESULTS: The cell proliferation of Arnica montana and Bellis perennis was evaluated by MTT assay. Osteogenic differentiation of C3H10T1/2 was induced by the addition of ß-glycerophosphate, ascorbic acid and dexamethasone in the differentiation medium over 3 weeks. Cells were treated with Arnica montana and Bellis perennis individually as well as in combination. The osteogenic differentiation potential of Arnica montana and Bellis perennis to differentiate C3H10T1/2 into osteoblasts was measured by alkaline phosphatase activity, alizarin red staining and the expression of Osteocalcin using immunostaining and qRT-PCR. Arnica montana and Bellis perennis could enhance C3H10T1/2 cell proliferation at 1600 µg. Further, the compound showed the ability to augment osteogenesis as confirmed by increased expression of alkaline phosphatase and enhanced calcium accumulation as seen by the Alizarin Red staining and quantification. Enhanced osteogenesis was further supported by the increased expression of osteocalcin in the treated cells with individual and combined doses of Arnica montana and Bellis perennis. Therefore, the findings provide additional support for the positive impact of Arnica montana and Bellis perennis on bone formation. CONCLUSIONS: Our findings suggest that homoeopathic compounds Arnica montana and Bellis perennis can augment osteogenesis individually as well as in combination.

Osteocalcin improves glucose tolerance, insulin sensitivity and secretion in older male mice.

Osteocalcin deficient mice (OC-/-), on a mixed 129/BL6J background, were reported to show glucose intolerance, insulin insensitivity and reduced insulin secretion at 1-6 mos of age. This is controversial as two studies in OC-/- mice on different backgrounds (C3H/BL6 (5-6 mos.) and C57BL/6N (5 and 9 mos.)) found no effect on glucose metabolism. To determine the role of OC in glucose metabolism we conducted glucose tolerance tests (GTT), insulin tolerances tests (ITT) and glucose stimulated insulin secretion (GSIS) on 6 and 9.5 month-old male OC-/- and OC+/+ mice on a pure C57BL/6J background and fed a normal chow diet. All results were analyzed with a two-way repeated measures ANOVA. The GTT results showed no effect on males at 6 months of age but glucose intolerance was significantly increased (p < 0.05) in male OC-/- mice at 9.5 months of age. The ITT results indicated significantly increased insulin resistance in male OC-/- mice. Glucose stimulated insulin secretion (GSIS) showed insulin significantly (p < 0.05) reduced in OC-/- at several time points. Mouse Osteocalcin injected into OC-/- mice decreased the glucose level. Our results confirm the role of OC in glucose metabolism and insulin sensitivity and demonstrate a role in insulin secretion in older male mice on a C57BL/6J background. Differences in background, age, or experimental procedures could explain controversial results. A delayed onset of the effect of OC on glucose metabolism at 9.5 months in male C57BL/6J mice highlights the importance of background on phenotype. Consideration of genetic background and age may be beneficial for human studies on osteocalcin and glucose homeostasis and may be relevant to the elderly where osteocalcin is reduced.

In vitro effects of uncarboxylated osteocalcin on buffalo Leydig cell steroidogenesis.

Uncarboxylated osteocalcin (UcOCN), a bone derived circulating protein, has been demonstrated to influence steroidogenesis in testicular Leydig cells of murine and human species. However, the role of UcOCN in testosterone biosynthesis remains unexplored in domestic animals. The present study aimed to investigate the impact of UcOCN on the expressions of steroidogenic genes (HSD3ß1, HSD3ß6, CYP17A1, CYP11A1), testosterone production and GPRC6A receptor localization in buffalo Leydig cells. Leydig cells from the testes of adult Murrah buffalo were isolated, with an average cell count and viability after digestion and Percoll enrichment of 1.43 × 106 cells/g of testes and 78.5%, respectively. Immunophenotyping of Percoll-enriched cell suspension by flow cytometry showed populations of Leydig cells ranging between 69 and 73.9%. Immunostaining confirmed the presence of GPRC6A receptors and CYP11A1 positive Leydig cells. When these cells were cultured and incubated with varying levels of UcOCN (6, 12, 24, and 48 ng/ml) and LH, there was a significant (P < 0.01) increase in testosterone production and up-regulation (P < 0.05) of CYP11A1, CYP17A1, HSD3ß1 and HSD3ß6 gene expression. In summary, the present study underscored the effects of UcOCN on testosterone biosynthesis, expression of crucial steroidogenic genes and interaction with GPRC6A receptors in buffalo Leydig cells, emphasizing its potential implications in andrology.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.

Histomorphometric and molecular characterization of stromal and mineralized components in fibro-osseous lesions.

BACKGROUND: Indistinct and analogous histopathological features of various fibro-osseous lesions make establishing a definitive diagnosis a challenge. There is a need for additional molecular and histochemical tools to support and differentiate these lesions in order to establish a concrete diagnosis. MATERIALS AND METHODS: A retrospective analysis of biopsied lesions in formalin-fixed paraffin-embedded sections (10 cases each of fibrous dysplasia, ossifying fibroma, and cement-osseous dysplasia) retrieved from the archives was studied for immunoexpression of osteocalcin (quantitative analysis in osteocytes), collagen characterization using Azan, Picrosirus, and Toluidine blue stain for evaluating intensity and localization of collagen fibers, and morphometric analysis of vasculature (for evaluating mean vessel density as square microns). RESULTS: Positive immunostaining of osteocalcin suggested mutations of the GNAS-1 gene found in fibrous dysplasia indirectly, as it is a negative regulator of bone formation. Osteocalcin immunopositivity was quantitatively measured in the fibro-osseous lesions, with fibrous dysplasia measuring 14.47 ± 3.628 as compared to ossifying fibroma measuring 5.23 ± 1.33, followed by cemento-osseous dysplasia measuring 2.30 ± 1.409. Toluidine blue suggests the presence of oxytalan fibers (resistant to acid hydrolysis) in ossifying fibroma and cemento-osseous dysplasia, pointing toward the pathogenesis of the lesion. Azan stain and Picrosirus (under a polarizing microscope) helped in distinguishing hard tissue characteristics (70% of cases of fibrous dysplasia showed only a magenta component followed by intermixed magenta with a blue component in 20% of cases and only 10% of cases showed magenta with blue borders whereas for ossifying fibroma, 40% of cases depicted magenta with blue borders along with the other 40% with intermixed magenta with blue component). The mean vessel density was also highest in fibrous dysplasia measuring 7.90 ± 1.079 (in Sq. micron area), followed by ossifying fibroma and cemento-osseous dysplasia. CONCLUSION: The diagnosis of fibro-osseous lesions by hematoxylin and eosin alone is confusing and thus should be supported by relatively simple histomorphometric analysis for better treatment outcomes. At the diagnostic stage of fibro-osseous lesions, evaluation of intralesional vessel size, reliable molecular marker, and histochemical nature can aid in differentiating fibrous dysplasia from central ossifying fibroma and cemento-osseous dysplasia alongside, other clinical, radiographic and pathological criteria. These parameters help in the diagnostic decision-making of fibro-osseous lesions.

Osteocalcin of maternal and embryonic origins synergize to establish homeostasis in offspring.

Many physiological osteocalcin-regulated functions are affected in adult offspring of mothers experiencing unhealthy pregnancy. Furthermore, osteocalcin signaling during gestation influences cognition and adrenal steroidogenesis in adult mice. Together these observations suggest that osteocalcin may broadly function during pregnancy to determine organismal homeostasis in adult mammals. To test this hypothesis, we analyzed in unchallenged wildtype and Osteocalcin-deficient, newborn and adult mice of various genotypes and origin maintained on different genetic backgrounds, the functions of osteocalcin in the pancreas, liver and testes and their molecular underpinnings. This analysis revealed that providing mothers are Osteocalcin-deficient, Osteocalcin haploinsufficiency in embryos hampers insulin secretion, liver gluconeogenesis, glucose homeostasis, testes steroidogenesis in adult offspring; inhibits cell proliferation in developing pancreatic islets and testes; and disrupts distinct programs of gene expression in these organs and in the brain. This study indicates that osteocalcin exerts dominant functions in most organs it influences. Furthermore, through their synergistic regulation of multiple physiological functions, osteocalcin of maternal and embryonic origins contributes to the establishment and maintenance of organismal homeostasis in newborn and adult offspring.

Osteocalcin: Promoter or Inhibitor of Hydroxyapatite Growth?

Osteocalcin is the most abundant noncollagenous bone protein and the functions in bone remineralization as well as in inhibition of bone growth have remained unclear. In this contribution, we explain the dual role of osteocalcin in the nucleation of new calcium phosphate during bone remodeling and in the inhibition of hydroxyapatite crystal growth at the molecular scale. The mechanism was derived using pH-resolved all-atom models for the protein, phosphate species, and hydroxyapatite, along with molecular dynamics simulations and experimental and clinical observations. Osteocalcin binds to (hkl) hydroxyapatite surfaces through multiple residues, identified in this work, and the fingerprint of binding residues varies as a function of the (hkl) crystal facet and pH value. On balance, the affinity of osteocalcin to hydroxyapatite slows down crystal growth. The unique tricalcium γ-carboxylglutamic acid (Gla) domain hereby rarely adsorbs to hydroxyapatite surfaces and faces instead toward the solution. The Gla domain enables prenucleation of calcium phosphate for new bone formation at a slightly acidic pH of 5. The growth of prenucleation clusters of calcium phosphate continues upon increase in pH value from 5 to 7 and is much less favorable, or not observed, on the native osteocalcin structure at and above neutral pH values of 7. The results provide mechanistic insight into the early stages of bone remodeling from the molecular scale, help inform mutations of osteocalcin to modify binding to apatites, support drug design, and guide toward potential cures for osteoporosis and hyperosteogeny.

Platelet-Rich Plasma Lysate Enhances the Osteogenic Differentiation of Adipose-Derived Stem Cells.

ABSTRACT: Adipose-derived stem cells (ADSCs) have become an accepted source of cells in bone tissue engineering. This study aimed to investigate whether platelet-rich plasma (PRP) lysate can replace traditional fetal bovine serum as a culture medium with the enhanced proliferation and osteogenic potential of ADSCs. We divided the experiment into 5 groups where the ADSCs were cultured in an osteogenic medium containing 2.5%, 5%, 7.5%, and 10% PRP lysate with 10% fetal bovine serum as the control group. The cell proliferation, alkaline phosphatase (ALP) activity, ALP stain, alizarin red stain, osteocalcin (OCN) protein expression, and osteogenic-specific gene expression were analyzed and compared among these groups. The outcome showed that all PRP lysate-treated groups had good ALP stain and ALP activity performance. Better alizarin red stains were found in the 2.5%, 5%, and 7.5% PRP lysate groups. The 2.5% and 5% PRP lysate groups showed superior results in OCN quantitative polymerase chain reaction, whereas the 5% and 7.5% PRP lysate groups showed higher OCN protein expressions. Early RUNX2 (Runt-related transcription factor 2 () genes were the most expressed in the 5% PRP lysate group, followed by the 2.5% PRP lysate group, and then the 7.5% PRP lysate group. Thus, we concluded that 5% PRP lysate seemed to provide the optimal effect on enhancing the osteogenic potential of ADSCs. Platelet-rich plasma lysate-treated ADSCs were considered to be a good cell source for application in treating nonunion or bone defects in the future.

Roles of vitamin K­dependent protein in biomineralization (Review).

Vitamin K (VK), a fat­soluble vitamin, is well known as an anticoagulant in the clinic. It is essential for the post­translational activation of VK­dependent proteins (VKDPs) because hydroquinone VK is a cofactor of glutamine carboxylase. At present, 17 VKDPs are known, which are mainly involved in coagulation and calcification. When Glu residues are carboxylated to Gla residues, these proteins gain a higher calcium­binding ability, which explains why VK has an important role in blood coagulation and biomineralization. However, the current view on the role of VK and several VKDPs in biomineralization remains inconsistent. For instance, conflicting results have been reported regarding the effect of osteocalcin gene knockout on the bone of mice; matrix Gla protein (MGP) promotes osteoblasts mineralization but inhibits vascular smooth muscle cell mineralization. The present review aimed to summarize the existing evidence that several VKDPs, including osteocalcin, MGP, Gla­rich protein and growth arrest specific 6 are closely related to calcification, including bone health, vascular calcification and lithiasis. The current review discussed these controversies and provided suggestions for future studies on VKDPs, i.e. taking into account dietary habits, geographical environments and genetic backgrounds.

Mechanosignaling-related angiocrine factors drive osteoblastic phenotype in response to zirconia.

BACKGROUND: The growing use of zirconia as a ceramic material in dentistry is attributed to its biocompatibility, mechanical properties, esthetic appearance, and reduced bacterial adhesion. These favorable properties make ceramic materials a viable alternative to commonly used titanium alloys. Mimicking the physiological properties of blood flow, particularly the mechanosignaling in endothelial cells (ECs), is crucial for enhancing our understanding of their role in the response to zirconia exposure. METHODS: In this study, EC cultures were subjected to shear stress while being exposed to zirconia for up to 3 days. The conditioned medium obtained from these cultures was then used to expose osteoblasts for a duration of 7 days. To investigate the effects of zirconia on osteoblasts, we examined the expression of genes associated with osteoblast differentiation, including Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Additionally, we assessed the impact of mechanosignaling-related angiocrine factors on extracellular matrix (ECM) remodeling by measuring the activities of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) during the acquisition of the osteogenic phenotype, which precedes mineralization. RESULTS: Our data revealed that mechanosignaling-related angiocrine factors play a crucial role in promoting an osteoblastic phenotype in response to zirconia exposure. Specifically, exposed osteoblasts exhibited significantly higher expression levels of genes associated with osteoblast differentiation, such as Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Furthermore, the activities of MMP2 and MMP9, which are involved in ECM remodeling, were modulated by mechanosignaling-related angiocrine factors. This modulation is likely an initial event preceding the mineralization phase. CONCLUSION: Based on our findings, we propose that mechanosignaling drives the release of angiocrine factors capable of modulating the osteogenic phenotype at the biointerface with zirconia. This process creates a microenvironment that promotes wound healing and osseointegration. Moreover, these results highlight the importance of considering the mechanosignaling of endothelial cells in the modulation of bone healing and osseointegration in the context of blood vessel effects. Our data provide new insights and open avenues for further investigation into the influence of mechanosignaling on bone healing and the osseointegration of dental devices.

The hyaluronic acid gel promotes the formation of osteoblasts mineralized nodules and fracture callus by regulating the expression of Runx2 and osteocalcin.

This study aimed to investigate the effect of hyaluronic acid (HA) gel regulating the expression of Runx2 and osteocalcin (OCN) on osteoblast mineralization nodules and fracture callus formation. To achieve this aim, MC3T3-E1 cells were divided into two groups, the HA group and the control group, based on the intervention of HA gel. In addition, a fracture callus model was constructed to observe cell proliferation, cell mineralization, and fracture callus formation. Results showed that HA at different concentrations had no obvious outcome on the proliferation of MC3T3-E1 cells (P>0.05). The area of mineralized nodules in the HA intervention group (65.38±4.27) was higher than in the control (9.52±2.16, P<0.05). The expression levels of Runx2 and OCN in the HA intervention group were higher than control (P<0.05). The callus area in the HA group (110.05±4.16) and (143.16±8.84) was significantly higher as against control (72.51±6.32, 88.92±5.28) at 2 and 4 weeks after intervention (P<0.05). It was concluded that HA gel promotes the proliferation and differentiation of osteoblasts by regulation of Runx2 and OCN, and then promotes the formation of mineralized nodules of osteoblasts and fracture callus, thereby promoting fracture healing.

Impact of history of periodontitis on gene expression of bone-related factors in young patients

Abstract Although dental implants and bone regenerative procedures are important approaches for the reestablishment of esthetics and function in young patients with a history of generalized aggressive periodontitis (GAP), no predictable outcomes have been reported, and the host osteo-immunoinflammatory response may play a relevant role in this context. In view of the lack of molecular investigations into the bone tissue condition of young patients with periodontitis, the aim of this study was to evaluate the gene expression of bone-related factors in this population. Bone biopsies were obtained from the posterior mandible in 16 individuals previously diagnosed with GAP and on periodontal support therapy and from 17 periodontally healthy (PH) patients. The gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), bone sialoprotein (BSP), and type I collagen (COL-I), important biomarkers of bone turnover, was evaluated by qRT-PCR. Lower TGF-β and OPG mRNA levels were observed in GAP patients compared to PH individuals (p ≤ 0.05). There were no between-group differences in levels of TNF-α, BSP, RANKL, OC, or COL-I mRNA (p>0.05). In young adults, a history of periodontal disease can negatively modulate the gene expression of important bone-related factors in alveolar bone tissue. These molecular outcomes may contribute to the future development of therapeutic approaches to benefit bone healing in young patients with history of periodontitis via modulation of osteo-immuno-inflammatory biomarkers.

Influence of low-level laser irradiation on osteocalcin protein and gene expression in bone tissue1

Abstract Purpose: To evaluate osteocalcin gene and protein expression in vitro and in an in vivo model of ostectomy. Methods: Twenty Wistar rats were assigned into two groups A (n=10, laser) and B (n=10, control). Ostectomy was performed in the femur diaphysis; the twenty fragments removed, composed in vitro groups named as in vivo (A and B) and cultivated in CO2 atmosphere for thirteen days. Low-level laser irradiation was performed in groups A (in vivo and in vitro) by an GaAlAs device (λ=808 nm, dose of 2J/cm2, power of 200mW, power density of 0.2W/cm2, total energy of 1.25J, spot diameter of 0.02mm) for 5 seconds, at one point, daily. It was performed immunocytochemistry assays in vivo and in vitro groups. In vitro groups were also submitted to RNA extraction, cDNA synthesis and gene expression by quantitative PCR. Statistical analysis was realized with p<0.05. Results: Immunocytochemistry scores showed no significant differences between control and laser groups either in vivo and in vitro. Gene expression also showed no statistical differences. Conclusion: Low-level laser irradiation did not alter osteocalcin protein and gene expression in vivo and in vitro in the studied period but it may have been expressed them in an earlier period.

Tridax procumbens flavonoids promote osteoblast differentiation and bone formation

BACKGROUND: Tridaxprocumbens flavonoids (TPFs) are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts. RESULTS: TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs) including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2. CONCLUSION: Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Use of calcium phosphate cement scaffolds for bone tissue engineering: in vitro study / A utilização do cimento de fosfato de cálcio como arcabouço para engenharia de tecido ósseo: estudo in vitro

Purpose: To evaluate the ability of macroporous tricalcium phosphate cement (CPC) scaffolds to enable the adhesion, proliferation, and differentiation of mesenchymal stem cells derived from human bone marrow. Methods: Cells from the iliac crest of an adult human donor were processed and cultured on macroporous CPC discs. Paraffin spheres sized between 100 and 250µm were used as porogens. Cells were cultured for 5, 10, and 15 days. Next, we assessed cells' behavior and morphology on the biomaterial by scanning electron microscopy. The expression levels of the BGLA and SSP1 genes and the alkaline phosphatase (ALP) activity were quantified by the quantitative real-time polymerase chain reaction technique (QT-PCR) using the fluorophore SYBR GREEN®. Results: QT-PCR detected the expression of the BGLA and SSP1 genes and the ALP activity in the periods of 10 and 15 days of culture. Thus, we found out that there was cell proliferation and differentiation in osteogenic cells. Conclusion: Macroporous CPC, with pore sized between 100 and 250µm and developed using paraffin spheres, enables adhesion, proliferation, and differentiation of mesenchymal stem cells in osteogenic cells and can be used as a scaffold for bone tissue engineering.

Objetivo: Avaliar a capacidade de suportes tridimensionais macroporosos de cimento de fosfato de cálcio (CFC), de permitir a adesão, proliferação e diferenciação de células-tronco mesenquimais derivadas da medula óssea humana. Métodos: células obtidas da crista ilíaca de um doador humano adulto foram processadas e cultivadas sobre suportes de CFC, macroporosos, que tiveram como corpo gerador de poros, microesferas de parafina, com tamanho entre 100 e 250µm. Os períodos de cultura estabelecidos foram de cinco, 10 e 15 dias. Após estes períodos, o comportamento e a morfologia das células junto ao biomaterial foram avaliados por meio de Microscopia Eletrônica de Varredura. Os níveis de expressão dos genes BGLA e SSP1 bem como a atividade da Fosfatase Alcalina (ALP) foram quantificados pela técnica de PCR em Tempo Real (QT-PCR) utilizando o fluoróforo SYBR Green®. Resultados: O QT-PCR detectou a expressão dos genes BGLA e SSP1 e a atividade da fosfatase alcalina nos períodos de 10 e 15 dias de cultura. No período de cinco dias, não foi observada a expressão de nenhum dos genes investigados. Conclusão: O CFC, macroporoso, com tamanho de poros entre 100 e 250µm, criados por meio da utilização de microesferas de parafina, permite a adesão, proliferação e diferenciação de células-tronco mesenquimais em células osteogênicas, podendo ser utilizado como arcabouço para engenharia de tecido ósseo.

Effects of low-level laser therapy on human osteoblastic cells grown on titanium

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.

Este estudo teve como objetivo investigar o efeito do laser diodo de gálio-alumínio-arsênio (GaAlAs) em células osteoblásticas humanas cultivadas sobre discos de Ti. Para tanto, células osteoblásticas foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sobre discos de Ti por 17 dias. As células foram submetidas à irradiação no 3º e 7º dias na dose de 3 J/cm2 e comprimento de onda de 780 nm e células não irradiadas foram usadas como controle. A irradiação não alterou a proliferação celular, atividade de ALP e formação de matriz mineralizada. Microscopia por epifluorescência indicou que após 24 h da aplicação do laser, as culturas irradiadas apresentaram áreas sem células, que mais tarde foram repovoadas por células em fase de proliferação e menos diferenciadas. O laser aumentou a expressão gênica relativa da ALP, OC, BSP e BMP-7 e reduziu a de RUNX2, OPN e OPG. Os resultados indicam que a terapia com laser modula de forma complexa as respostas celulares, estimulando a diferenciação osteoblástica. Assim, é possível sugerir possíveis benefícios do laser na osseointegração de implantes de Ti apesar do efeito deletério às células imediatamente após a irradiação.